The BDs from human BRD2 (BD1: 65–194; BD2: 347–455), BRD3 (BD1: 25–147; BD2: 307–419), and BRD4 (BD1: 42–168; BD2: 348–464) were expressed in Escherichia coli from pQE80L-Navi (Qiagen) as N-terminally His-tagged and biotinylated proteins for SPR. For other experiments, they were expressed from pGEX-6P (Cytiva) as N-terminal glutathione-S-transferase-fusion proteins. Expression and purification, via affinity chromatography and size-exclusion chromatography, of unlabeled and uniformly 15N-labeled proteins were carried out as described previously (11 (link)).
Pgex 6p
Manufactured by Cytiva
The PGEX-6P is a plasmid vector used for the expression and purification of recombinant proteins in Escherichia coli. It contains the tac promoter for inducible expression and a protease cleavage site for the removal of fusion tags.
Automatically generated - may contain errors
2 protocols using pgex 6p
1
Expression and Purification of BRD Proteins
2
Recombinant Expression and Purification of KIAA0513
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We cloned a sequence encoding cDNA of the isoform c of human KIAA0513 (accession number: NP_001284695.1) into pGEX-6P (Cytiva, Pittsburgh, PA). We induced expression of the cDNA product by treating Escherichia coli (E. coli) KRX (Promega, Madison, WI) cells harboring the pGEX-6P-KIAA0513 and pMINOR with 0.5 mM isopropyl-β-D-thiogalactoside at 37℃ for 3 h [39] (link). The cells were lysed by sonication in phosphate-buffered saline containing 2 mM dithiothreitol. The proteins were puri ed using Glutathione-Sepharose 4 Fast Flow medium (Cytiva) and HiPrep 26/10 Desalting column (Cytiva). Puri ed glutathione S-transferase (GST)-KIAA0513 was concentrated to 3.5 mg/ml in phosphate-buffered saline containing 2 mM dithiothreitol.
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